Process for preparing optically active 1,2-diols

ABSTRACT

The present invention relates to a process for producing optically active diols represented by the general formula (II):                    
     wherein R 1  represents (CH 2 ) n , CH═CH, O, S or NH whereupon n is an integer of 1 to 4, and R 2  represents hydrogen, C 1-6  alkyl, C 1-6  alkoxy, (C 1-6  alkoxy)-carbonyl, hydroxy, carboxy, halogen, nitro or amino, which comprises treating compounds represented by the general formula (I):                    
     wherein R 1  and R 2  have the same meanings as defined above, with a culture of a microorganism belonging to the genus Rhodococcus, Bacillus, Brevibacterium or Gordona and being capable of stereoselectively diolating a double bond in ring A, or with a culture of  Mortierella vinacea , or with said microorganism itself, or with a treated material from said microorganism.

TECHNICAL FIELD

The present invention relates to a process for producing opticallyactive 1,2-diols.

The optically active 1,2-diols are useful as various pharmaceuticalpreparations and optically active, biologically active substances, aswell as intermediates of their derivatives.

For example, cis-(1S,2R)-dihydroxyindane ortrans-(1R,2R)-dihydroxyindane can be an important starting material ofCrixivan used as an anti-AIDS drug because of its inhibitor activity onproteolytic enzymes for AIDS virus (J. Med. Chem., 34, 1228 (1991)).

BACKGROUND ART

Known methods of microbially producing optically active 1,2-diolsinclude, for example, a method of reacting indene along with a culturedmicroorganism of the genus Pseudomonas (J. Chem. Soc., Chem. Commun.,339 (1989); J. Chem. Soc., Chem. Commun., 117 (1995)), a method ofadding a derivative such as indene, 1,2-dihydronaphthalene or the liketo a culture of Mortierella isabellina to convert it into a hydroxide(Bioorg. Med. Chem., 2, 439 (1994)), a method of using as a startingmaterial optically active 2-bromo-1-hydroxyindane generated uponasymmetrical reduction of 2-bromoindane-1-one with Cryptococcus macerans(J. Org. Chem., 43, 4540 (1978)), and a method of stereoselectivelyhydrolyzing 1-methoxy-2-acetoxyindane in a culture of Rhizopus nigricans(J. Org. Chem., 49, 675 (1984)).

Further, known methods of synthesizing optically active1,2-dihydroxyindane derivatives by chemical synthesis include a methodof reacting 2-bromo-1-hydroxyindane in the presence of acetic acid oracetic acid and water (J. Chem. Soc. Perkin Trans. I., 2767 (1982)) anda method of oxidatively hydrating indene (Synthesis, 1142 (1985)).

However, these conventional methods, particularly chemical syntheticmethods, have the problems that the starting materials are expensive,the operation is cumbersome and the yield is low because of themultiple-step reaction, or the optical purity of the resulting1,2-dihydroxyindane is low, while the microbial methods also have theproblem that the range of utilizable microorganisms is limited.

DISCLOSURE OF THE INVENTION

The object of the present invention is to provide a process forproducing optically active 1,2-diols efficiently by microorganisms notused in the conventional microbial methods.

That is, the present invention encompasses:

1. A process for producing optically active diols represented by thegeneral formula (II):

wherein R¹ represents (CH₂)_(n), CH═CH, O, S or NH whereupon n is aninteger of 1 to 4, and R² represents hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,C₁₋₆ alkoxy-carbonyl, hydroxy, carboxy, halogen, nitro or amino, whichcomprises treating compounds represented by the general formula (I):

wherein R¹ and R² have the same meanings as defined above, with aculture of a microorganism belonging to the genus Rhodococcus, Bacillus,Brevibacterium or Gordona and being capable of stereoselectivelydiolating a double bond in ring A, or with said microorganism itself, orwith a treated material from said microorganism.

2. The process according to item 1 above wherein the microorganismcapable of stereoselectively diolating a double bond in ring A is amicroorganism belonging to the genus Rhodococcus.

3. The process according to item 2 above wherein the microorganismbelonging to the genus Rhodococcus is Rhodococcus rhodochrous ATCC21199.

4. The process according to item 2 above wherein the microorganismbelonging to the genus Rhodococcus is Rhodococcus rhodochrous ATCC21198.

5. The process according to item 1 above wherein the microorganismcapable of stereoselectively diolating a double bond in ring A is amicroorganism belonging to the genus Bacillus.

6. The process according to item 1 above wherein the microorganismcapable of stereoselectively diolating a double bond in ring A is amicroorganism belonging to the genus Brevibacterium.

7. The process according to item 1 above wherein the microorganismcapable of stereoselectively diolating a double bond in ring A is amicroorganism belonging to the genus Gordona.

8. The process according to item 1 above wherein substrate-adsorptivecarriers are added to the reaction solution.

9. A process for producing optically active diols represented by thegeneral formula (II):

wherein R¹ represents (CH₂)_(n), CH═CH, O, S or NH whereupon n is aninteger of 1 to 4, and R² represents hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,(C₁₋₆ alkoxy)-carbonyl, hydroxy, carboxy, halogen, nitro or amino, whichcomprises treating compounds represented by the general formula (I):

wherein R¹ and R² have the same meanings as defined above, with aculture of Mortierella vinacea, or with said microorganism itself, orwith a treated material from said microorganism.

10. The process according to item 9 above wherein substrate-adsorptivecarriers are added to the reaction solution.

In the definition of each group in the compounds shown in the generalformula (I) or (II), the alkyl moiety in C₁₋₆ alkyl, C₁₋₆ alkoxy and(C₁₋₆ alkoxy)-carbonyl may be straight-chain or branched insofar as itis alkyl containing 1 to 6 carbon atoms, and examples of such alkylinclude methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl,pentyl and hexyl. The halogen refers to each atom of fluorine, chlorine,bromine or iodine.

The purity of the compounds represented by the general formula (I),which are used in the present invention, is not particularly limited,and those of high purity or low purity may be used. Impurities arepreferably compounds not adversely affecting the growth and existence ofthe microorganism and the enzyme activity, but there is no particularlimit insofar as their influence is negligible.

The compounds represented by the general formula (I) are preferablythose wherein R¹ is (CH₂)_(n), more preferably those wherein R¹ is CH₂or (CH₂)₂.

The microorganisms used in the present invention may be anymicroorganisms belonging to the genus Rhodococcus, Bacillus,Brevibacterium or Gordona, or Mortierella vinacea.

The microorganisms belonging to the genus Rhodococcus include e.g.Rhodococcus rhodochrous ATCC 21198, Rhodococcus rhodochrous ATCC 21199,Rhodococcus ruber JCM 3205, Rhodococcus sp. IFM 18 and Rhodococcusgloberulus ATCC 25714.

The microorganisms belonging to the genus Bacillus include e.g. Bacillusmegaterium IAM 1032 and Bacillus pasteurii ATCC 11859.

The microorganisms belonging to the genus Brevibacterium include e.g.Brevibacterium acetylicum ATCC 953.

The microorganisms belonging to the genus Gordana include e.g., Gordanarubropertinctus ATCC 27863 (equivalent to Rhodococcus ruber JCM 3205).

Mortierella vinacea includes e.g. Mortierella vinacea TKBC 1102.

Rhodococcus rhodochrous ATCC 21198, Rhodococcus rhodochrous ATCC 21199,Rodococcus globerulus ATCC 25714, Bacillus pasteurii ATCC 11859,Brevibacterium acetylicum ATCC 953 and Gordona rubropertinctus ATCC27863 are stored in American Type Culture Collection (ATCC) in 12301Parklawn Drive, Rockville, Md. 20852, U.S.A. and also appear in acatalogue.

Rhodococcus ruber JCM 3205 is stored in Japan Collection ofMicroorganisms (JCM) in the Institute of Physical and Chemical Research(RIKEN) in Hirosawa 2-1, Wako-shi, Saitama, JP and also appears in acatalogue.

Rhodococcus sp. IFM 18 is stored in Research Center for Pathogenic Fungi& Microbiol Toxicosis, Chiba University (formerly Institute of FoodMicrobiology) in Inohana 1-8-1, Chuo-ku, Chiba-shi, Chiba, JP and alsoappears in a catalogue.

Bacillus megaterium IAM 1032 is stored in IAM Culture Collection (IAM),Center for Cellular and Molecular Research, Institute of Molecular andCellular Biosciences, the University of Tokyo in Yayoi 1-1-1, Bunkyo-ku,Tokyo, JP and also appears in a catalogue.

Mortierella vinacea TKBC 1102 is stored in TKBC Culture Collection(TKBC), Institute of Biological Science, University of Tsukuba inTennodai 1-1-1, Tsukuba-shi, Ibaraki, JP and also appears in acatalogue.

Among the microorganisms described above, preferable microorganismsinclude microorganisms belonging to the genus Rhodococcus, particularlyRhodococcus rhodochrous such as Rhodococcus rhodochrous ATCC 21198 andRhodococcus rhodochrous ATCC 21199.

In the present invention, the compounds of the general formula (I) aboveare treated with a culture (e.g. a culture liquid) of the microorganism,the microorganism itself or a treated material from the microorganism(e.g. a material from the disrupted microorganism, an extract from themicroorganism, or a crude or purified enzyme from the microorganism).

As the form of this treatment reaction, mention is made of e.g. a methodof directly adding the substrate, that is, the compound of the generalformula (I) above, to a culture liquid of the growing or grownmicroorganism, a method of suspending the isolated grown microorganismin a buffer and then adding the substrate thereto, and a method ofimmobilizing the microorganism in a usual manner, then floating it in astirring chamber or charging it into a column, and adding the substratethereto.

The substrate may be added all at once, but preferably added inportions. Alternatively, a continuous reaction method of continuouslyadding the substrate while continuously recovering the product can alsobe used.

Usually, the composition of the medium for culturing the microorganismused in the present invention may be any suitable medium in which themicroorganism can grow, but the following composition can be mentionedas a preferable example. That is, the carbon source is glucose, mannitolor a mixture thereof, and the nitrogen source is yeast extract, cornsteep liquor, peptone, meat extract or a mixture thereof. Further,inorganic materials such as common salt and manganese sulfate arepreferably added.

Culture of the microorganism used in the present invention may beconducted in a usual manner, for example at pH 5 to 9, preferably 6 to8, at a culture temperature of 20 to 40° C., preferably 25 to 30° C.,and aerobically for 24 to 72 hours, but when the reaction is conductedby adding the substrate to the culture liquid, culture may be continuedfor 300 hours or more.

When the reaction is conducted by adding the substrate to the cultureliquid, it is preferable that oil such as soybean oil or silicon oil isadded at a concentration of 5 to 50%, preferably 10 to 20%, or a carriercapable of adsorbing the substrate, for example adsorptive resin (e.g.HP-20 resin, Mitsubishi Chemical) or dried yeast (Ebios™, Asahi BeerYakuhin), is added at a concentration of about 0.5 to 10%, preferably 1to 5%, in order to prevent the vaporization of the substrate or to avoidthe inhibition of growth by the substrate.

When the microorganism is reacted after harvested, it is preferable touse the microorganism grown by adding the compound of the generalformula (I) or its reduced compound such as indene, indane, naphthaleneor dihydronaphthalene as an inducer for enzyme activity during culture.

Culture is conducted preferably at a reaction temperature of 15 to 50°C., preferably 20 to 30° C. and at pH 5 to 10, preferably 6 to 8. Thesubstrate may be added all at once, but preferably added in portions,and when added to the medium, the substrate is adjusted to aconcentration in the range of 0.02 to 1.0%, after which it can be addedlittle by little as the reaction proceeds. Further, in the case of thereaction with the microorganism or with the immobilized microorganism,the concentration of the substrate is regulated in the range of 0.1 to1.0%, after which the substrate can be added little by little as thereaction proceeds. The reaction is conducted usually under shaking orstirring. Although the reaction time varies depending on theconcentration of the substrate, the density of the microorganism and anyother reaction conditions, the reaction time is preferably selected suchthat the reaction in the case where the substrate is added to theculture medium is finished in 100 to 300 hours or more, or the reactionwith the microorganism or with the immobilized microorganism is finishedin 6 to 72 hours. In respect of yield, it is preferable that while thereaction is analyzed by taking an aliquot of the reaction solution, thereaction is terminated when the reaction stops.

The reaction with the microorganism isolated after culture or with theimmobilized microorganism may be conducted in a sealed vessel in whichan air layer corresponding to a stoichiometrically necessary amount ofoxygen is kept to prevent the vaporization of the substrate.

To recover the thus obtained optically active 1,2-diol from the reactionsolution, general techniques, for example extraction with organicsolvent such as ethyl acetate or isopropyl acetate, can be used. Forthis recovery, the microorganism may be removed by centrifugation orfiltration as necessary prior to extraction of the desired product.Alternatively, after removal of the microorganism from the reactionsolution, the desired product may be recovered by passing the solutionthrough a column packed with suitable adsorptive resin (e.g. SP207resin, Mitsubishi Chemical) and then eluting the adsorbed product with asuitable solvent such as acetonitrile and methanol.

The crude extract thus obtained is dehydrated over anhydrous sodiumsulfate etc., and the solvent is removed under reduced pressure wherebycrude optically active 1,2-diol can be obtained. It can be furtherpurified by recrystallization after suspended in a solvent such as ethylacetate or through various kinds of chromatography.

BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLE 1

A medium, pH 7.0, containing 4% mannitol, 5% dried yeast (Ebios™, AsahiBeer Yakuhin), 0.005% manganese sulfate and 0.25% defoaming agentAdekanol LG109™ (Asahi Denka Kogyo K. K.) was prepared in de-ionizedwater, and 2.5 L of the medium was introduced into 5-L jar fermenter andsterilized at 120° C. for 20 minutes in an autoclave.

Rhodococcus rhodochrous ATCC 21199 was inoculated at a density of 2.4%into the medium, while 0.2% indene was added thereto, and indene dilutedat a concentration of 66% with ethanol was added continuously thereto.The culture temperature was set at 30° C., stirring at 550 rpm, andaeration at 0.5 vvm. The rate of addition of the substrate wascontrolled by periodically taking an aliquot of the reaction solutionand measuring it for the concentration of indene by high performanceliquid chromatography (HPLC), whereby the concentration of indene in themedium was adjusted to 0.2%. The concentration of indene was measured byHPLC (column, Shim-pack CLC-ODS (Shimadzu Corporation); solvent,acetonitrile/water=4/1, 40° C., 1 ml/min., 248 nm).

After 143 hours, an aliquot of the reaction solution was removed andmeasured by HPLC (column, Chiral-pack AD (Daicel Ltd.); solvent,hexane/ethanol=92/8, 40° C., 0.5 ml/min., 220 nm), indicating that 2.20g/l trans-(1R,2R)-indanediol, 0.06 g/l cis-(1S,2R)-indanediol and 1.00g/l 2-hydroxy-1-indanone had been accumulated. The added indene was 11.2g/l in total.

After 143 hours, the culture was suspended, and the microorganism wasremoved from the culture liquid by centrifugation. The supernatant wasapplied to a column packed with about 150 g adsorptive resin SP207(Mitsubishi Chemical), washed with water and eluted with acetonitrile.The crude extract thus obtained was dehydrated over anhydrous sodiumsulfate, concentrated, dissolved in ethyl acetate at 70° C. andcrystallized at a gradually decreasing temperature to givetrans-(1R,2R)-indanediol. This crystal was further recrystallized fromethyl acetate to give 2.6 g crystal.

The optical purity of the resulting optically active indanediol wasmeasured by HPLC (column, Chiralcell OK (Daicel Ltd.); solvent,hexane/isopropanol=95/5, 40° C., 0.5 ml/min., 220 nm), indicating thatit was almost 100% e.e. trans-(1R,2R)-indanediol.

Measurement of its optical rotation indicated that [α]_(D) was−28.7°(c=0.936, ethanol).

EXAMPLE 2

A medium, pH 7.0, containing 1% peptone, 0.7% meat extract, 0.5% yeastextract and 0.3% common salt was prepared in de-ionized water, and 1.9 Lof the medium was introduced into a 5-L jar fermenter and sterilized at120° C. for 20 minutes in an autoclave. Thereafter, 150 g/300 ml glucoseseparately sterilized in an autoclave was added thereto.

Rhodococcus rhodochrous ATCC 21199 was inoculated at a density of 2.4%into the medium and cultured in the same manner as in Example 1.

After 24 hours, about 25 g HP-20 resin (Mitsubishi Chemical) suspendedin 400 ml de-ionized water and 6.25 ml defoaming agent Adekanol LG109(Asahi Denka Kogyo K. K.) were added thereto, and further 1 ml indenewas added thereto.

Thereafter, culture was continued while 1 ml indene was added at 8-hourintervals.

After 212 hours, the culture was suspended and the concentration of theproduct was determined by HPLC, indicating that 5.87 g/ltrans-(1R,2R)-indanediol had been accumulated. The optical purify ofthis trans-isomer was almost 100% e.e. in chromatography.

EXAMPLE 3

A medium, pH 7.0, containing 3% corn steep liquor, 1% yeast extract and3% glucose was prepared in de-ionized water, and 10 ml of the medium wasintroduced into a thick test tube and sterilized at 120° C. for 15minutes in an autoclave.

Rhodococcus rhodochrous ATCC 21199 was inoculated via a loop of platinuminto the medium and cultured for 49.5 hours under shaking. Thereafter,0.01 ml indene was added thereto, and the culture was continued foradditional 16 hours.

Then, the concentration of the product was determined by HPLC,indicating that 0.55 g/l trans-(1R,2R)-indanediol, 0.15 g/lcis-(1S,2R)-indanediol and 0.19 g/l 2-hydroxy-1-indanone had beenaccumulated. The optical purity of the trans-isomer was almost 100% e.e.in chromatography.

EXAMPLE 4

A medium, pH 7.0, containing 1% peptone, 0.7% meat extract, 0.5% yeastextract, 0.3% common salt and 3% glucose was prepared in de-ionizedwater, and 15 L of the medium was introduced into a 30-L jar fermenterand sterilized at 120° C. for 30 minutes.

7.5 g naphthalene dissolved in 15 ml dimethylformamide was added to themedium, and Rhodococcus rhodochrous ATCC 21199 was inoculated at adensity of 3% into the medium and cultured at a temperature set at 30°C., stirring at 300 rpm and aeration at 0.5 vvm. After 32.25 hours, 150g/300 ml glucose was added thereto, and the culture was continued. After78 hours, the culture liquid was centrifuged to give 483 g microbialpellet. 0.2 g of this microbial pellet was placed in a 15 ml plastictube and suspended in 1 ml of 0.1 M potassium phosphate buffer, pH 8.0.0.003 ml indene was added thereto, and the tube was sealed and incubatedat 28° C. for 18 hours. After reaction, the product was extracted with 1ml ethyl acetate and analyzed by HPLC, indicating that 2.0 g/ltrans-(1R,2R)-indanediol, 0.02 g/l cis-(1S,2R)-indanediol and 0.91 g/l2-hydroxy-1-indanone had been formed. The optical purify of thetrans-isomer was almost 100% e.e. in chromatography.

EXAMPLE 5

10 g of the microbial pellet obtained in Example 4 was suspended byadding 10 ml water, and 1.5 g κ-carrageenan dissolved in 40 ml water wasadded thereto and well mixed therewith. This mixture was charged into aninjection cylinder and dropped little by little into 1 L of 0.3 Maqueous potassium chloride to give 42 g granular immobilizedmicroorganism.

1 g of this immobilized microorganism was introduced into a 15 mlplastic tube and added to 2 ml of 0.1 M Tris-HCl buffer, pH 8.0, and0.03 ml indene was added thereto, and the tube was sealed and incubatedat 28° C. for 18.5 hours.

1 ml supernatant, which was obtained by centrifugation after reaction,was extracted with 2 ml ethyl acetate and analyzed by HPLC, indicatingthat 0.42 g/l trans-(1R,2R)-indanediol, 0.05 g/l cis-(1S,2R)-indanedioland 0.12 g/l 2-hydroxy-1-indanone had been formed. The optical purity ofthe trans-isomer was almost 100% e.e. in chromatography.

EXAMPLE 6

20 g of the microbial pellet obtained in Example 4 was suspended byadding 20 ml water, and 3 g sodium alginate dissolved in 80 ml water wasadded thereto and well mixed therewith. This mixture was charged into aninjection cylinder and dropped little by little into 1.5 L of 0.1 Maqueous calcium chloride to give 98 g granular immobilizedmicroorganism.

This immobilized microorganism was incubated and measured in the samemanner as in Example 5. The result indicated that 0.55 g/ltrans-(1R,2R)-indanediol, 0.08 g/l cis-(1S,2R)-indanediol and 0.14 g/l2-hydroxy-1-indanone had been formed. The optical purity of thetrans-isomer was almost 100% e.e. in chromatography.

EXAMPLE 7

0.5 g/l trans-(1R,2R)-dihydroxytetrahydronaphthalene was formed in thesame operation as in Example 3 except that dihydronaphthalene was usedin place of indene used in Example 3.

EXAMPLE 8

10 ml of a medium, pH 7.0, containing 1% peptone, 0.7% meat extract,0.5% yeast extract, 0.3% common salt, 1% glucose and 0.25% defoamingagent Adekanol LG109 (Asahi Denka Kogyo K.K.) was introduced into athick test tube and sterilized at 120° C. for 15 minutes in anautoclave.

Each strain shown in Table 1 was inoculated into the medium and culturedat 28° C. under shaking. After 19 hours, 0.03 ml indene was addedthereto, and the shake culture was further continued. After 40 hours intotal, the reaction was terminated, and the product was extracted withethyl acetate and analyzed. The results are shown in Table 1.

TABLE 1 Optical Puri- Steric Strain ty (% e.e.) ConfigurationRhodococcus rhodochrous ATCC 21198 100 1S, 2R Rhodococcus sp. IFM 18 931R, 2S Rhodococcus globerulus ATCC 25714 45 1R, 2S Gordonarubropertinctus ATCC 27863 93 1R, 2S Bacillus megaterium IAM 1032 13 1S,2R Bacillus pasteurii ATCC 11859 7 1S, 2R Brevibacterium acetylicum ATCC953 100 1S, 2R

EXAMPLE 9

10 ml medium, pH 7.0, containing 2% malt extract, 2% glucose and 0.1%peptone was introduced into a thick test tube and sterilized at 120° C.for 15 minutes in an autoclave.

The strain shown in Table 2 was inoculated into the medium and culturedat 25° C. under shaking. After 68 hours, 0.03 ml indene was addedthereto, and the shake culture was further continued. The reaction wasterminated after 89 hours in total, and the product was extracted withethyl acetate and analyzed. The result is shown in Table 2.

TABLE 2 Optical Strain Purity (% e.e.) Steric Configuration Mortierellavinacea TKBC 1102 35 1S, 2R

INDUSTRIAL APPLICABILITY

The optically active 1,2-diols produced according to the process of thepresent invention are useful as various pharmaceutical preparations,optically active, biologically active substances, and intermediates oftheir derivatives.

What is claimed is:
 1. A process for producing an optically active diolof the formula (II):

wherein R¹ is (CH₂)_(n), CH═CH, O, S or NH whereupon n is an integer of1 to 4, and R² is hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, (C₁₋₆alkoxy)-carbonyl, hydroxy, carboxy, halogen, nitro or amino, whichcomprises treating a compound of the formula (I):

wherein R¹ and R² have the same meanings as defined above, with aculture of a microorganism belonging to the genus Rhodococcus, Bacillus,or Brevibacterium and being capable of stereoselectively diolating adouble bond in ring A, or with said microorganism itself, or with acrude or purified enzyme from said microorganism; and recovering theoptically active diol; wherein the microorganism belonging to the genusRhodococcus is selected from the group consisting of Rhodococcusrhodochrous ATCC 21198, Rhodococcus rhodochrous ATCC 21199, Rhodococcusruber JCM 3205, Rhodococcus sp. IFM 18, and Rhodococcus globerulus ATCC25714; and wherein the microorganism belonging to the genus Bacillus isselected from the group consisting of Bacillus megaterium IAM 1032 andBacillus pasteurii ATCC
 11859. 2. The process according to claim 1wherein the microorganism capable of stereoselectively diolating adouble bond in ring A is a microorganism belonging to the genusRhodococcus and is selected from the group consisting of Rhodococcusrhodochrous ATCC 21198, Rhodococcus rhodochrous ATCC 21199, Rhodococcusruber JCM 3205, Rhodococcus sp. IFM 18, and Rhodococcus globerulus ATCC25714.
 3. The process according to claim 2 wherein the microorganismbelonging to the genus Rhodococcus is Rhodococcus rhodochrous ATCC21199.
 4. The process according to claim 2 wherein the microorganismbelonging to the genus Rhodococcus is Rhodococcus rhodochrous ATCC21198.
 5. The process according to claim 1 wherein the microorganismcapable of stereoselectively diolating a double bond in ring A is amicroorganism belonging to the genus Bacillus and is selected from thegroup consisting of Bacillus megaterium IAM 1032 and Bacillus pasteuriiATCC
 11859. 6. The process according to claim 1 wherein themicroorganism capable of stereoselectively diolating a double bond inring A is a microorganism belonging to the genus Brevibacterium.
 7. Theprocess according to claim 1 wherein substrate-adsorptive carriers areadded to the reaction solution.
 8. A process for producing an opticallyactive diol of the formula (II):

wherein R¹ is (CH₂)_(n), CH═CH, O, S or NH whereupon n is an integer of1 to 4, and R² is hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, (C₁₋₆alkoxy)-carbonyl, hydroxy, carboxy, halogen, nitro or amino, whichcomprises treating a compound of the formula (I):

wherein R¹ and R² have the same meanings as defined above, with aculture of Mortierella vinacea, or with said microorganism itself, orwith a crude or purified enzyme from said microorganism; and recoveringthe optically active diol.
 9. The process according to claim 8 whereinsubstrate-adsorptive carriers are added to the reaction solution. 10.The process according to claim 6, wherein the microorganism belonging tothe genus Brevibacterium is Brevibacterium acetylicum ATCC
 953. 11. Theprocess according to claim 1, wherein the compound of formula (I) isindene.
 12. The process according to claim 2, wherein the compound offormula (I) is indene.
 13. The process according to claim 3, wherein thecompound of formula (I) is indene and the diol of formula (II) istrans-(1R,2R)-indanediol.
 14. The process according to claim 4, whereinthe compound of formula (I) is indene and the diol of formula (II) iscis-(1S,2R)-indanediol.
 15. The process according to claim 9, whereinthe compound of formula (I) is indene.